The existence of peptides in the atrium which are responsible for maintenance of normal extracellular fluid parameters--i.e., the volume and pressure of liquid in the blood vessels--is well known. A series of closely related peptides, designated atrial natriuretic peptides, have been isolated from several species and identified, and analogs of these peptides have been prepared.
The natriuretic effect of a crude extract of rat atrial tissue was demonstrated over seven years ago. A number of peptides with diuretic and natriuretic properties have since been isolated from atrial tissue and sequenced: Flynn, T.G., et al., Biochem Biophys Res Commun (1983) 117:859-865; Currie, M.G., et al., Science (1984) 223:67-69; Kangawa, K., et al., Biochem Biophys Res Commun (1984) 118:131-139; U.S. Pat. No. 4,496,544; U.S. Pat. No. 4,508,712; Kangawa, K., et al., Biochem Biophys Res Commun (1984) 119:933-940; Garcia, R., et al., Biochem Biophys Res Commun (1985) 126:178-174; Katsube, N., et al., Biochem Biophys Res Commun (1985) 128:325-330; U.S. Pat. Nos. 4,607,023; 4,557,864; and 4,618,600; copending applications 616,488, now abandoned; 766,030, now U.S. Pat. Nos. 4,764,504; and 870,795. These peptides, called atrial natriuretic peptides (ANPs), are cyclic disulfides comprising 17 amino acids in the cycle (including the two cysteines which provide the disulfide bond). The gene which encodes them encodes a larger protein which is then processed into shorter versions which make up the set of ANPs.
Various analogs of the isolated atrial peptides are also described in copending application Ser. Nos.921,360, now abandoned; 138,893; now abandoned; and 174,739now U.S. Pat. No. 4,935,492.
It is understood that these peptides and their analogs are effective in regulating blood pressure by controlling fluid volume and vessel diameter. A number of disease states are characterized by abnormal fluid retention, including congestive heart failure, cirrhosis of the liver, and nephrotic syndrome. These diseases are associated with excessive fluid accumulation on the venous side of circulation, and an underperfusion of the kidneys, leading to a fall in glomerular filtration rate (GFR). In addition, reduced renal perfusion stimulates secretion of renin, a proteolytic enzyme which, in the circulation, leads to the formation of angiotensin, a powerful constrictor of the arteriole. Renin also stimulates release of the sodium-retaining hormone aldosterone by the adrenal gland.
Hypertension per se is another serious result of an increase in extracellular fluid volume and is a major cause of death.
Therapeutic measures related to diseases associated with sodium and water retention are varied and include administration of a variety of diuretic substances. However, no single therapeutic agent is satisfactory for all individuals, and it is important to enhance the repertoire of available materials. The present invention provides additional materials which, besides their supplementation of the repertoire of useful therapeutics, are important in that they are found in brain and atrium and thus may shed light on the central and peripheral mechanisms whereby normal individuals maintain the appropriate fluid balance. In addition, some of these peptides and proteins have modified and altered physiological activities.
One of these factors from porcine brain has been isolated and sequenced by Sudoh, P., Nature (1988) 332:78-81. It is a 26-amino acid peptide synthesized in porcine brain and atrial tissue. In atrial tissue, the peptide is present at about 1/100 of the concentration of analyzed atrial natriuretic peptide (ANP) activity. However, in brain the peptide may be more abundant than ANP. The spectrum of activity of this porcine brain natriuretic peptide, or pBNP, is similar to that of the porcine ANP. A comparison of the amino acid sequences of a portion of human ANP (hANP) and the pBNP is shown below; the corresponding relevant portion of the porcine ANP is identical to the human sequence ##STR4##
There are nine (starred) positions which are not homologous. The conservative substitution of Leu for Ile or Met, found in rat or human ANP sequences, respectively, is a known acceptable substitution.
Subsequent papers from this same group at Miyazaki Medical College further characterize these proteins. Sudoh, T., et al., Biochem Biophys Res Comm (1988) 155:726-732, report the isolation of a 32-amino acid natriuretic peptide ("BNP-32") from porcine brain which contains the 26 amino acids of the porcine BNP described above at its C-terminus and an additional N-terminal 6-amino acid extended portion of the sequence Ser-Pro-Lys-Thr-Met-Arg-. In papers following on subsequent pages, levels of various natriuretic peptides in tissues are reported. Ueda, S., et al., (ibid.), pp. 733-739, utilized a radioimmunoassay to localize and measure the levels of porcine BNP and porcine BNP-32 in the brain and spinal cord. The results showed that both BNP and BNP-32 were major forms of immunoreactive BNP in the porcine brain, and that the highest concentrations were found in the medulla-pons, striatum, and spinal cord. The porcine form of atrial natriuretic peptide (pANP) was also found in the porcine brain but at a level approximately 13 times lower than that characteristic of BNP. Minamino, N., et al. (ibid.), pp. 740-746, report the results of radioimmunoassay for porcine BNP and ANP in peripheral tissue. The concentration of BNP was highest in cardiac atrium of the tissues assayed. The immunoreactive form of this protein was characterized as mostly a 12 kd high molecular weight form; less than 15% of the total immunoreactive BNP in atrial tissue is of the lower molecular weight forms pBNP or pBNP-32.
In a subsequent issue of this publication, Minamino, N., et al., Biochem Biophys Res Comm (1988) 157:402-409, reported the isolation and characterization of this higher molecular weight form of BNP from porcine heart. The complete amino acid sequence of this protein was obtained and shown to contain the 26-amino acid pBNP (and 32-amino acid pBNP-32) at its carboxy terminus. The full-length protein contains 106 amino acids. Finally, Maekawa, K., et al. (ibid.), pp. 410-416, report the cloning and sequence analysis of a cDNA. encoding a precursor protein for porcine BNP. A cDNA library was obtained from porcine cardiac atrium and the relevant BNP-encoding gene was isolated and sequenced. The gene was found to include a 25-residue putative signal peptide at the N-terminus followed by the codons corresponding to the 106 amino acids of the reported protein.
These results are consistent with the information available from studies of the atrial-derived natriuretic peptides which are generally also associated with longer precursors. In the parent application herein, the gene encoding porcine BNP was provided, which permitted the putative amino acid sequence of the upstream portion of these precursor proteins to be deduced. While the cDNA obtained in the parent application was incompletely processed and contained an intron, further manipulation of this sequence using standard techniques as described below permitted the location of the intron to be established. The parent application disclosed a straightforward procedure which could be applied to identify natriuretic-related peptides (NRP) from other species. The direct procedure outlined did not permit cloning of human NRP. As will be discussed further below, it was not possible to obtain human NRP using only porcine BNP as a probe. It was necessary to use an intermediary clone, canine BNP, to obtain human NRP. Accordingly, the invention provides a family of natriuretic peptides (NPs) and natriuretic-related peptides (NRPs) from a variety of vertebrate source.